We will characterize structural determinants of HIV-1 Vif, HIV-2 Vif, A3G, and A3F that interact and induce degradation of A3G and A3F. The determinants of HIV-1 Vif that interact with A3G and A3F are not conserved in HIV-2 Vif, suggesting that HIV-2 Vif induces A3 protein degradation using distinct interactions. We will carry out extensive mutational analysis of HIV-1 Vif, HIV-2 Vif, A3G, and A3F to identify structural determinants of each protein that interact with each other. We will also characterize determinants of A3G-A3G interactions in cells using bimolecular fluorescence complementation. We will develop small-molecule inhibitors of A3G-Vif and A3F-Vif interactions. We have developed in situ assays for high-throughput screening of small-molecule inhibitors that interfere with HIV-1 Vif-mediated degradation of A3G and A3F. In collaboration with the NIH Chemical Genomics Center, we will screen a library of &gt;300,000 compounds to identify inhibitors of this novel target for antiviral drug development. We will also identify inhibitors of HIV-2 Vif and A3 interaction for the treatment of HIV-2 infection. We will determine the impact of A3G- and A3F-mediated hypermutation on the HIV-1 genome. To gain insight into the impact of hypermutation on viral evolution, we will carry out ultradeep sequencing analysis to identify and characterize the entire HIV-1 genome with respect to A3-mediated G-to-A hypermutation target sites to identify mutational hotspots and coldspots. We will identify the site of ubiquitination on A3F. We will quantify the antiviral activity of A3 proteins in primary CD4+ T cells and macrophages. We will determine the relative inhibitory potential of A3G and A3F in primary activated CD4+ T cells and macrophages using different modes of activation and cytokine stimulation to gain insights into their antiviral activity under various physiological conditions. We will analyze the role of A3 proteins in P bodies, and the mechanism by which Mov10 inhibits retroviral replication. We have observed that Mov10, an interferon-inducible P body protein, inhibits HIV-1 replication at the level of virus production and reverse transcription. To elucidate the mechanism of inhibition, we will analyze Mov10 virion incorporation and its effects on Gag expression and reverse transcription. To gain insights into the interactions of APOBEC3 proteins with viral components after infection, we will use live-cell imaging to determine the kinetics with which APOBEC3 proteins dissociate from the viral preintegration complexes. [Corresponds to Pathak Project 2 in the October 2011 site visit report of the HIV Drug Resistance Program]